This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. As regulation by ubiquitin modification plays a role in nearly every cellular pathway, it is critical to identify sites of ubiquitin attachment. These sites have been identified by mass spectrometry based on the increase in molecular mass of a tryptic peptide carrying two additional glycine residues from the ubiquitin moiety. However, such peptides with GG shifts have been difficult to discover. We devised a method to enrich for GG-peptides prior to mass spectrometry by two steps. First, before affinity purification of ubiquitin, we reduce the complexity of the proteome by treatment with 2-nitro-5-thiocyanobenzoic acid (NTCB);NTCB cleaves at cysteine residues and ubiquitin contains no cysteine. Second, we separate the highly charged GG-peptides by strong cation exchange (SCX) chromatography.